High sensitivity of asymmetric 18F-THK5351 PET abnormality in patients with corticobasal syndrome

Corticobasal syndrome (CBS) is characterized by symptoms related to the asymmetric involvement of the cerebral cortex and basal ganglia. However, early detection of asymmetric imaging abnormalities can be challenging. Previous studies reported asymmetric 18F-THK5351 PET abnormalities in CBS patients, but the sensitivity for detecting such abnormalities in larger patient samples, including early-stage cases, remains unclear. Patients clinically diagnosed with CBS were recruited. All patients displayed asymmetric symptoms in the cerebral cortex and basal ganglia. Asymmetric THK5351 PET abnormalities were determined through visual assessment. Brain MRI, perfusion SPECT, and dopamine transporter (DAT) SPECT results were retrospectively reviewed. The 15 patients had a median age of 72 years (59–86 years) and a disease duration of 2 years (0.5–7 years). Four patients met the probable and 11 met the possible CBS criteria according to Armstrong criteria at the time of PET examination. All patients, including early-stage cases, exhibited asymmetric tracer uptake contralateral to their symptom-dominant side in the cerebral cortex/subcortical white matter and striatum (100%). The sensitivity for detecting asymmetric imaging abnormalities contralateral to the symptom-dominant side was 86.7% for brain MRI, 81.8% for perfusion SPECT, and 90% for DAT SPECT. White matter volume reduction was observed in the subcortical region of the precentral gyrus with increased THK5351 uptake, occurring significantly more frequently than gray matter volume reduction. THK5351 PET may be a sensitive imaging technique for detecting asymmetric CBS pathologies, including those in early stages.

by MAO-B inhibitors on average (five mild cognitive impairment, two AD dementia, and one PSP), leading to the creation of second-generation tau tracers with reduced off-target binding 9 . In PSP, THK5351 uptake was reduced by MAO-B inhibitors completely in an autoradiography study 12 and 69-89% in an in vivo human study 14 , suggesting that most tracer uptake was due to MAO-B. Physiologically, MAO-B is abundant in the striatum, thalamus, and brainstem 15,16 ; pathologically, it is present in high concentrations in reactive astrocytes at astrogliosis lesion sites 17 . Although 11 C-l-Deprenyl PET has been utilized to detect MAO-B/astrogliosis, 18 F-THK5351 PET provides enhanced sensitivity 18 and is now applied as a PET ligand for detecting astrogliosis in various neurological conditions [19][20][21][22][23][24][25][26][27] .
In previous research, Kikuchi et al. found asymmetric 18 F-THK5351 PET tracer uptake in brain regions that corresponded to symptoms in five CBS patients who fulfilled modified Cambridge criteria 28 and had a disease duration between 1.5 and 4.6 years 29 . This group later reported longitudinal data from five CBS patients and proposed that 18 F-THK5351 PET could also be valuable for disease monitoring, predominantly reflecting astrogliosis 30 . Moreover, they observed a higher precentral gyrus tracer accumulation in seven CBS patients compared to PSP, AD, and healthy controls 31 . A single case study also revealed that 18 F-THK5351 PET could detect astrogliosis in an affected brain region of a patient with CBD presenting with frontal behavioral-spatial syndrome (FBS) 32 . While these findings suggest that 18 F-THK5351 PET has potential as a diagnostic and disease-monitoring biomarker, its application in CBS patients is limited, and its efficacy in detecting asymmetric abnormalities in a larger patient population, including those in earlier stages, is yet to be determined. Furthermore, no comparisons have been made between 18 F-THK5351 PET results and those of other imaging tests.
In this study, we present the findings of 18 F-THK5351 PET and additional imaging techniques in an expanded cohort of CBS patients, encompassing individuals in the early stages of the disease.

Methods
Subjects. Patients diagnosed with CBS were enrolled for the THK5351 PET study from January 2020 to March 2023. We conducted a retrospective review of the participants' medical records and included those with a clinical diagnosis of CBS as of February 2023. All patients exhibited progressively worsening asymmetric higher cortical and extrapyramidal symptoms, meeting either probable or possible CBS according to the Armstrong criteria 1 at the time of PET examinations.
PET image acquisition and analysis. 18 F-THK5351 was prepared at the Tokyo Metropolitan Institute for Geriatrics and Gerontology, and PET imaging followed a previously established protocol 16 . PET images were normalized using the cerebellar cortex as a reference region, with the uptake set to one (uptake ratio index [URI]). Images were visually assessed, and asymmetry was evaluated by two specialists who were blinded to the clinical information and interpreted the brain PET images (K. Ishibashi and K. Ishii). The tracer uptake in the striatum was quantified by Dr. View/LINUX version R2.0 (AJS, Tokyo, Japan) by setting symmetric region of interest (ROI) (M. Kurihara).
Image acquisition and analysis of other images. The results of brain structural MRI, perfusion single photon emission tomography (SPECT) using 123 I-IMP or 99m Tc-ECD, and dopamine transporter (DAT) SPECT using 123 I-FP-CIT were retrospectively reviewed. The results of statistical analyses were available for all SPECT tests (eZIS or 3D-SSP for perfusion SPECT and DAT View for DAT SPECT). Gray and white matter volume reduction analyses (voxel-based morphometry) based on 3D T1-weighted images using SPM8 plus DARTEL using Voxel-based Specific Regional Analysis system for Alzheimer's Disease (VSRAD) advance 2 software 33 were unavailable in five patients with CBS. The asymmetry of the MRI and SPECT findings was evaluated visually with reference to these statistical analyses, when available. These evaluations were conducted by trained radiologists (A.M. Tokumaru and M. Kameyama) who were blinded to the PET results.
Determination of amyloid positivity. For those who consented, amyloid PET imaging using [ 11 C] Pittsburgh compound B (PiB) PET 34 or cerebrospinal fluid (CSF) biomarker testing using ELISA 35 was employed to estimate amyloid positivity, as reported previously. In cases with discordant results, amyloid PET emission tomography results were used. Statistical methods. Statistical analyses were conducted using the software program R version 4.0.3 (R Foundation for Statistical Computing, Vienna, Austria) and graphical interface EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) 36 , or GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA). Missing data were addressed using a pairwise deletion approach. Categorical variables were expressed as percentages, and differences between groups were assessed using Fisher's exact test. Continuous variables were displayed as medians (full range). Statistical significance was set at p < 0.05. This

Results
Baseline characteristics.  28 ; however, the remaining patients did not meet the criteria at the time of PET examination due to an insufficient number of criteria met (n = 5) or unavailability of trials to rule out L-dopa responsiveness (n = 1). Twelve patients were amyloid-negative, and three were amyloid-positive. All patients underwent THK5351 PET and brain structural MRI, while brain perfusion SPECT and DAT SPECT were performed in 11 and 10 patients, respectively. MRI volume reduction analysis results were available for 10 of the 15 patients, and was unavailable in 5 due to lack of 3D T1-weighted images.
Concordance of asymmetric imaging abnormalities with symptom dominant sides. Representative 18 F-THK5351 PET images are provided in Fig. 1. All CBS patients exhibited asymmetric THK5351 uptake in the cerebral cortex and subcortical white matter, aligning with their symptoms. Asymmetric uptake was also observed in the striatum, corresponding to the laterality of symptoms in all patients ( Supplementary  Fig. 1). The concordance of the laterality of imaging abnormalities (contralateral to the symptom-dominant side) was 100% for THK5351 PET in both the cerebral cortex/subcortical white matter and striatum, 86.7% for brain structural MRI, 81.8% for brain perfusion SPECT, and 90% for DAT SPECT (Fig. 2). Limb kinetic apraxia, ideomotor apraxia, cortical sensory deficits, and speech or language impairments were observed in 13, 10, 7, and 10 patients, respectively. THK5351 uptake was detected in the corresponding brain regions of most patients ( Table 2).
Association between THK5351 uptake and volume reduction of gray and white matter at the precentral gyrus analyzed by MRI. THK5351 PET images overlaid on structural brain MRI images indicated that THK5351 uptake occurred in both the cerebral cortex and associated white matter (Fig. 1). Previous research has proposed that THK5351 uptake in the precentral gyrus is specific to CBS 31 , while we have previously reported that MRI white matter volume reduction analysis using SPM8 plus DARTEL is effective in detecting abnormalities in the frontal subcortical white matter around the precentral gyrus in CBS patients 37 . Thus, we assessed the relationship between THK5351 uptake and brain volume reduction around the precentral gyrus in patients with available 3D T1-weighted MRI images (n = 10). Images can be found in Fig. 3 and Supplementary  Fig. 2.
Increased THK5351 uptake was observed in the right precentral gyrus in 6 patients (60%) and in the left precentral gyrus in 6 patients (60%). In these 12 precentral gyri, subcortical white matter volume reduction was suggested in 11 (91.7%) gyri, which was significantly more frequent than the gray matter volume reduction indicated in these gyri (3 [25%]; p = 0.00276).

Discussion
We conducted THK5351 PET in 15 patients with CBS, including those in early stages. THK5351 PET identified asymmetric abnormalities contralateral to the symptom-dominant side with high sensitivity in both the cerebral cortex/subcortical white matter and the striatum.
The high sensitivity of asymmetric THK5351 PET abnormalities may contribute to early diagnosis of CBS. The diagnosis of CBS is based on clinical findings, and several diagnostic criteria have been proposed 1,28,38 . Although these diagnostic criteria are useful for diagnosis at advanced stages, their sensitivity may be insufficient in early stages 28 . The diagnostic criteria proposed in 2013 by Armstrong et al. included two sets of criteria for CBS with different diagnostic certainties: probable and possible 39 . Although using possible CBS criteria may aid in earlier diagnosis, its specificity is low, and incorporating imaging biomarkers may be necessary 39 .
Compared to other imaging techniques, THK5351 PET has several advantages. First, THK5351 PET exhibited a clear asymmetric abnormality recognizable through visual assessment, without the need for complex statistical image analysis. Second, THK5351 PET detected asymmetric abnormalities with higher concordance with (contralateral to) the laterality of the symptom-dominant sides compared to other imaging techniques. Third, THK 5351 PET identified asymmetric abnormalities in both the cerebral cortex/subcortical white matter and striatum, while other imaging techniques detected in either the cerebral cortex/subcortical white matter or striatum. Although still in research use and not specific for tau pathology, these characteristics may warrant future studies on THK5351 PET for the early diagnosis of CBS.
Another finding of this study was the correlation between THK5351 uptake in the precentral gyrus and white matter volume reduction in the subcortical region of the precentral gyrus, rather than gray matter volume reduction, as suggested by MRI voxel morphometry. CBD and atypical presentations of PSP (PSP-CBS) may display MRI signal abnormalities in the white matter corresponding to the disease pathology at autopsy [40][41][42] . White matter volume reduction analyses can reveal volume reductions in the frontal subcortical white matter around the precentral gyrus in patients with CBS 37 and in other brain regions with other presentations of CBD 10 . A previous case report of a patient with CBD-FBS also suggested that the distribution of increased THK5351 uptake and white matter volume reduction were closely correlated in the frontal lobe, with reactive astrocytes observed along the corticomedullary junction in this brain region at autopsy 32   www.nature.com/scientificreports/ white matter volume reduction correlate in the subcortical region of the precentral gyrus in patients with CBS, although further studies with neuropathological assessments are needed to clarify the detailed distribution correlation between THK5351 and related pathologies (tau aggregates and astrogliosis) in both gray and white matter. Astrogliosis is observed in affected brain regions across a wide range of neurodegenerative diseases. Recent evidence suggests that it may not be a simple downstream phenomenon of neurodegeneration but may partially contribute to disease progression in some diseases 43 . Although next-generation tau tracers specific for tau should have the advantage of visualizing the distribution of aggregated tau itself, combining the evaluation of the regional  19,20 . Moreover, astrogliosis imaging may have the potential for disease monitoring, in combination with cerebrospinal and plasma GFAP concentrations, in future studies assessing disease-modifying therapies. The neuropathological backgrounds of our patients with CBS are undetermined at this point. Previous autopsy studies have shown that patients with CBS have underlying pathology of CBD, PSP, AD, or rarely FTLD-TDP 2,3 . While twelve patients with CSF or amyloid PET results suggesting negative amyloid pathology likely have CBD or PSP, the underlying pathology of three with results suggesting positive amyloid pathology are difficult to determine. Case 1 showed slightly decreased CSF Aβ42 without memory impairment or increased uptake in medial temporal lobe. We assume that this patient has non-AD CBS with or without incidental amyloid pathology. Case 4 showed severely decreased CSF Aβ42 and the distribution of THK-5351 uptake was different from others ( Fig. 1M-P) including medial and lateral temporal lobe. We assume that this patient has CBS-AD. Case 15 showed severely decreased CSF Aβ42 and increased CSF p-tau181. The patient showed memory impairment and increased uptake in medial temporal lobes, although lesions in the neocortex did not include lesions characteristic of CBS-AD such as lateral temporal lobes. We assume that this patient has non-AD CBS and mild cognitive impairment due to AD, although the possibility of CBS-AD remains.
This study has several limitations. First, neuropathological diagnosis was not available. Second, the sample size was small. Third, although previous studies have suggested that the majority of the increased THK5351 uptake represents MAO-B in non-AD patients [12][13][14] , and we excluded patients with AD by biomarkers in most patients, THK5351 uptake may represent both MAO-B and tau aggregates in some patients, especially in those with amyloid positivity. Future studies using a modified tracer 18 F-SMBT-1 specific for MAO-B 44 may resolve Figure 2. Concordance of imaging abnormality laterality against symptoms. The percentages of patients with asymmetric imaging abnormalities contralateral to the symptom-dominant side (concordant) are shown in blue. *Although brain MRI was available for all 15 patients, volumetric analyses based on 3D T1-weighted images were not available for 5 patients. Volumetric analyses were available for one patient with no laterality (CBS with CSF biomarker suggesting AD) and were unavailable for one patient with discordant results. THK 18 F-THK5351 PET, MRI structural magnetic resonance imaging, SPECT brain perfusion single-photon emission tomography, DAT dopamine transporter SPECT. www.nature.com/scientificreports/ this issue. Fourth, while not only laterality but also the regions with increased THK5351 uptake corresponded to those expected from clinical symptoms, these concordances were not perfect. Follow-up studies are warranted to evaluate whether increased THK5351 uptake in these lesions will be observed later on.
In conclusion, THK5351 PET may be a sensitive imaging technique for detecting asymmetric CBS pathologies, including those in early stages.

Data availability
The data sets and full protocol of the present study are available from the corresponding author on reasonable request.